The objectives of the proposed research are to elucidate the mechanism of methemoglobin reduction as it occurs in erythrocytes, to probe the role of proteases in the origin of the components of the methemoglobin reduction system, and to establish the structure of the prosthetic group and the biological activity of an erythrocyte green hemeprotein. We will study the chemical and physical properties of the two forms of bovine erythrocyte cytochrome b5, and compare these proteins with bovine liver microsomal cytochrome b5. The nature of the blocked N-terminal residue and the sites(s) of carbohydrate attachment will be investigated. We will attempted to detect, isolate, and characterize erythrocyte and reticulocyte proteases which will convert microsomal forms of cytochrome b5 and cytochrome b5 reductase to the erythrocyte forms of these proteins. The kinetics of methemoglobin reduction will be studied in intact cells, in hemolysates, and in a system reconstituted from purified cytochrome b5, cytochrome b5 reductase, methemoglobin, and NADH or NADPH. We will study the system in erythrocytes from normal individuals and from patients with congenital methemoglobinemia or congenital sensitivity to oxidant drugs. We will search for the biological activity of the bovine erythrocyte green hemeprotein. We will determine the molecular weight of the prosthetic group of this protein, derivatize this heme, and characterize these derivatives spectrally.